Conformational Dynamics of Single pre-mRNA Molecules During \u3cem\u3eIn Vitro\u3c/em\u3e Splicing

The spliceosome is a complex small nuclear RNA (snRNA)-protein machine that removes introns from pre-mRNAs via two successive phosphoryl transfer reactions. The chemical steps are isoenergetic, yet splicing requires at least eight RNA-dependent ATPases responsible for substantial conformational rearrangements. To comprehensively monitor pre-mRNA conformational dynamics, we developed a strategy for single-molecule FRET (smFRET) that uses a small, efficiently spliced yeast pre-mRNA, Ubc4, in which donor and acceptor fluorophores are placed in the exons adjacent to the 5′ and 3′ splice sites. During splicing in vitro, we observed a multitude of generally reversible time-and ATP-dependent conformational transitions of individual pre-mRNAs. The conformational dynamics of branchpoint and 3′-splice site mutants differ from one another and from wild type. Because all transitions are reversible, spliceosome assembly appears to be occurring close to thermal equilibrium

Molecular modelling of the GIR1 branching ribozyme gives new insight into evolution of structurally related ribozymes

Twin-ribozyme introns contain a branching ribozyme (GIR1) followed by a homing endonuclease (HE) encoding sequence embedded in a peripheral domain of a group I splicing ribozyme (GIR2). GIR1 catalyses the formation of a lariat with 3 nt in the loop, which caps the HE mRNA. GIR1 is structurally related to group I ribozymes raising the question about how two closely related ribozymes can carry out very different reactions. Modelling of GIR1 based on new biochemical and mutational data shows an extended substrate domain containing a GoU pair distinct from the nucleophilic residue that dock onto a catalytic core showing a different topology from that of group I ribozymes. The differences include a core J8/7 region that has been reduced and is complemented by residues from the pre-lariat fold. These findings provide the basis for an evolutionary mechanism that accounts for the change from group I splicing ribozyme to the branching GIR1 architecture. Such an evolutionary mechanism can be applied to other large RNAs such as the ribonuclease P

Single Molecule Analysis Research Tool (SMART): An Integrated Approach for Analyzing Single Molecule Data

Single molecule studies have expanded rapidly over the past decade and have the ability to provide an unprecedented level of understanding of biological systems. A common challenge upon introduction of novel, data-rich approaches is the management, processing, and analysis of the complex data sets that are generated. We provide a standardized approach for analyzing these data in the freely available software package SMART: Single Molecule Analysis Research Tool. SMART provides a format for organizing and easily accessing single molecule data, a general hidden Markov modeling algorithm for fitting an array of possible models specified by the user, a standardized data structure and graphical user interfaces to streamline the analysis and visualization of data. This approach guides experimental design, facilitating acquisition of the maximal information from single molecule experiments. SMART also provides a standardized format to allow dissemination of single molecule data and transparency in the analysis of reported data

Understanding the Origins of Bacterial Resistance to Aminoglycosides through Molecular Dynamics Mutational Study of the Ribosomal A-Site

Paromomycin is an aminoglycosidic antibiotic that targets the RNA of the bacterial small ribosomal subunit. It binds in the A-site, which is one of the three tRNA binding sites, and affects translational fidelity by stabilizing two adenines (A1492 and A1493) in the flipped-out state. Experiments have shown that various mutations in the A-site result in bacterial resistance to aminoglycosides. In this study, we performed multiple molecular dynamics simulations of the mutated A-site RNA fragment in explicit solvent to analyze changes in the physicochemical features of the A-site that were introduced by substitutions of specific bases. The simulations were conducted for free RNA and in complex with paromomycin. We found that the specific mutations affect the shape and dynamics of the binding cleft as well as significantly alter its electrostatic properties. The most pronounced changes were observed in the U1406C∶U1495A mutant, where important hydrogen bonds between the RNA and paromomycin were disrupted. The present study aims to clarify the underlying physicochemical mechanisms of bacterial resistance to aminoglycosides due to target mutations

MÉTODOS DE INDEXAÇÃO DE INDICADORES NA AVALIAÇÃO DA QUALIDADE DO SOLO EM RELAÇÃO À EROSÃO HÍDRICA

A avaliação da qualidade dos solos agrícolas é importante para definição e adoção de práticas de manejo que garantam a sustentabilidade socioeconômica e ambiental. Os métodos de indexação dos indicadores de qualidade denominados Índice de Qualidade Integrado (IQI) e Índice de Qualidade Nemoro (IQN) foram utilizados neste estudo para avaliar a qualidade de solo em áreas experimentais de plantio de eucalipto. A seleção dos indicadores foi feita a partir de nove indicadores de qualidade do solo: diâmetro médio geométrico, permeabilidade à água, matéria orgânica, macro e microporosidade, volume total de poros, densidade do solo, resistência à penetração e índice de floculação, que estão relacionados à erosão hídrica. Os tratamentos constituíram de eucalipto plantado em nível, com e sem a manutenção dos resíduos, em desnível e solo descoberto, em dois biomas distintos, cujas vegetações nativas são Cerrado e Floresta. Os índices de qualidade do solo (IQS) apresentaram alta correlação com a erosão hídrica. Entre os sistemas manejados, o Eucalipto com manutenção do resíduo evidenciou valores mais elevados em ambos os índices, ressaltando-se a importância da cobertura vegetal e manutenção da matéria orgânica para conservação do solo e da água em sistemas florestais. Os IQS demonstraram alto coeficiente de correlação inversa com as perdas de solo e água. Em locais com as maiores taxas de erosão hídrica manifestaram também os menores valores de IQI e IQN. Assim, os índices testados permitiram avaliar com eficácia os efeitos dos manejos adotados sobre a qualidade do solo em relação à erosão hídrica

A rugged free energy landscape separates multiple functional RNA folds throughout denaturation.

The dynamic mechanisms by which RNAs acquire biologically functional structures are of increasing importance to the rapidly expanding fields of RNA therapeutics and biotechnology. Large energy barriers separating misfolded and functional states arising from alternate base pairing are a well-appreciated characteristic of RNA. In contrast, it is typically assumed that functionally folded RNA occupies a single native basin of attraction that is free of deeply dividing energy barriers (ergodic hypothesis). This assumption is widely used as an implicit basis to interpret experimental ensemble-averaged data. Here, we develop an experimental approach to isolate persistent sub-populations of a small RNA enzyme and show by single molecule fluorescence resonance energy transfer (smFRET), biochemical probing and high-resolution mass spectrometry that commitment to one of several catalytically active folds occurs unexpectedly high on the RNA folding energy landscape, resulting in partially irreversible folding. Our experiments reveal the retention of molecular heterogeneity following the complete loss of all native secondary and tertiary structure. Our results demonstrate a surprising longevity of molecular heterogeneity and advance our current understanding beyond that of non-functional misfolds of RNA kinetically trapped on a rugged folding-free energy landscape

Fused Lasso for Feature Selection using Structural Information

Feature selection has been proven a powerful preprocessing step for high-dimensional data analysis. However, most state-of-the-art methods suffer from two major drawbacks. First, they usually overlook the structural correlation information between pairwise samples, which may encapsulate useful information for refining the performance of feature selection. Second, they usually consider candidate feature relevancy equivalent to selected feature relevancy, and some less relevant features may be misinterpreted as salient features. To overcome these issues, we propose a new fused lasso for feature selection using structural information. Our idea is based on converting the original vectorial features into structure-based feature graph representations to incorporate structural relationship between samples, and defining a new evaluation measure to compute the joint significance of pairwise feature combinations in relation to the target feature graph. Furthermore, we formulate the corresponding feature subset selection problem into a least square regression model associated with a fused lasso regularizer to simultaneously maximize the joint relevancy and minimize the redundancy of the selected features. To effectively solve the challenging optimization problem, an iterative algorithm is developed to identify the most discriminative features. Experiments demonstrate the effectiveness of the proposed approach

BOBA FRET: Bootstrap-based analysis of single-molecule FRET data

Time-binned single-molecule Förster resonance energy transfer (smFRET) experiments with surface-tethered nucleic acids or proteins permit to follow folding and catalysis of single molecules in real-time. Due to the intrinsically low signal-to-noise ratio (SNR) in smFRET time traces, research over the past years has focused on the development of new methods to extract discrete states (conformations) from noisy data. However, limited observation time typically leads to pronounced cross-sample variability, i.e., single molecules display differences in the relative population of states and the corresponding conversion rates. Quantification of cross-sample variability is necessary to perform statistical testing in order to assess whether changes observed in response to an experimental parameter (metal ion concentration, the presence of a ligand, etc.) are significant. However, such hypothesis testing has been disregarded to date, precluding robust biological interpretation. Here, we address this problem by a bootstrap-based approach to estimate the experimental variability. Simulated time traces are presented to assess the robustness of the algorithm in conjunction with approaches commonly used in thermodynamic and kinetic analysis of time-binned smFRET data. Furthermore, a pair of functionally important sequences derived from the self-cleaving group II intron Sc.ai5γ (d3'EBS1*/IBS1*) is used as a model system. Through statistical hypothesis testing, divalent metal ions are shown to have a statistically significant effect on both thermodynamic and kinetic aspects of their interaction. The Matlab source code used for analysis (bootstrap-based analysis of smFRET data, BOBA FRET), as well as a graphical user interface, is available via http://www.aci.uzh.ch/rna/